DNA double strand breaks (DSBs) are caused by endogenous (byproducts of cellular metabolism and replication associated errors) and exogenous (ionizing radiation and chemotherapeutic drugs) agents. Unrepaired or misrepaired DSBs can result in senescence, inducted apoptosis, or chromosomal aberrations, including translocations and deletions. These chromosomal aberrations can lead to genomic instability and tumorigenesis. To counteract the effects of DNA DSBs, two highly efficient DSB repair pathways have evolved in eukaryotic cells: non-homologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ pathway utilizes several enzymes, including Ku70/80 and DNA-PKcs, that capture both DNA ends, bring them together in a synaptic complex, and facilitate direct ligation of the DSB. HR is initiated once 5'-3' resection of the DSB occurs. The 5'-3' resection creates ssDNA ends which are subsequently used for strand invasion and exchange into a homologous DNA template and once resolved, the DSB is fully repaired. One of the major unresolved questions in the field of DNA repair is the mechanism that modulates the pathway choice between NHEJ and HR. The goal of this proposal is to test our hypothesis that the binding of Ku70/80 to DSBs plays a key role in protecting DNA ends regardless of the cell cycle stage and that dissociation of Ku from DSBs is one of the mechanisms responsible for modulating pathway choice between NHEJ and HR. In G1, we hypothesize that Ku70/80 mediates NHEJ-mediated DSB repair, but in S/G2 phases of the cell cycle it protects DNA from non-specific end processing until it is actively displaced from DSB ends to allow DNA end resection and HRmediated DSB repair. To test this hypothesis, we propose the following specific aims: 1) To test the hypothesis that Ku70/80 is required for DNA end stability in S phase of the cell cycle. Furthermore, we will determine if Ku70/80 blocks DNA end processing by assessing its ability to block DNA end resection via the known human factors responsible for this process using model DNA substrates, including a mononucleosome chromatin substrate in vitro. 2) To further support our hypothesis that Ku70/80 must be displaced from DSB ends for DNA end resection and HR to initiate, we will test if these processes are attenuated if DSB ends are persistently occupied by Ku in vivo. 3) To determine the mechanism that modulates Ku70/80's dissociation from DSBs to allow the initiation of DNA end resection and HR in S/G2 phases of the cell cycle. Basic mechanistic insights into DSB repair mechanisms and the regulation of pathway choice for the repair of DSBs is important as DSB repair is paramount for protecting the human genome. This is supported by the observations that an increase in cancer frequency is observed in mice and humans with mutations in genes that encode proteins responsible for the repair of DSBs. Furthermore, induction of DSBs is used as a therapeutic modality for cancer treatment. Taken together, these underlie the importance of understanding the coordination and function of DSB repair and insights into repair mechanisms will ultimately translate into clinical targets and benefits.